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Application Note Channel: CaV1.2Cells: CHOTools: SyncroPatch® 384PE

The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and neuroendocrine regulation1. VGCCs are composed of 4 subunits, which include an alpha 1 subunit which forms the Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs2 and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions1. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p133. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes4. In addition, in the light of the CiPA initiative, the L-type channel is likely to become an important target for cardiac safety testing5,6.

Here we present high quality data with reliable pharmacology on CaV1.2 expressing CHO cells at a high throughput collected on the SyncroPatch® 384PE. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range7-11 and success rates of >70% for completed experiments were recorded. Importantly, CaV1.2 recorded on the Syncro-Patch 384PE exhibited stable peak amplitudes during the course of the experiment and displayed little or no run-down.

Stability and Pharmacology of CaV1.2 Channels on Nanion’s SyncroPatch® 384PEThe electrophysiology team at Nanion Technologies GmbH, Munich.Cells kindly provided by Charles River.

Summary ResultsFor the evaluation of the performance of CHO-CaV1.2 cells, seal resistance values were determined from one experiment (Fig. 1) at the start and end of the experiment. Experiments were performed in perforated patch mode using multi-hole (4 holes per well) chips and seal resistance values were calculated and displayed per hole.

Figure 1: Statistics of CaV1.2 expressing CHO cells recorded on one NPC-384 chip on the SyncroPatch® 384PE. Success rate (seal resistance) of individual CHO cells on the SyncroPatch® 384. Shown is a bar graph of seal resistances at the start (light blue) and end of the experiment (dark blue). Multi-hole (4X) chips were used and the seal resistance values per hole are displayed.

start of experiment end of experiment

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phone +49 89 218997972fax +49 89 218997960www.nanion.de • info@nanion.de

Application NoteCurrents mediated by CaV1.2 could be reliably recorded on the SyncroPatch® 384PE with a high success rate (>70% for completed experiments). Figure 2 shows a screenshot of the data acquisition and analysis soft-ware of the SyncroPatch® 384PE during an experiment recording the current-voltage relationship of CaV1.2 expressed in CHO cells. The current-voltage plot for an average of 272 cells is shown in Panel C. A Boltzmann equation reveals a V0.5 of activation of -4.8 mV with a reversal potential of 57 mV in good agreement with the literature for L-type Ca2+ channels recorded from human ventricular myocytes12-14.

Figure 2: Typical recording of CaV1.2 expressed in CHO cells on the SyncroPatch® 384PE. A The screenshot shows the data acquisition and analysis software used on the SyncroPatch® 384PE. Three hundred and eighty-four small color-coded pictures shown in the upper left part from A1 to P24 display 384 recordings. Wells are color-coded based on seal resistance. One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. The online analysis values are shown for a current-voltage experiment. B The raw traces from an example cell elicited by depolarizing steps from -60 mV to 40 mV in 10 mV increments from a holding potential of -80 mV are shown. The black trace highlights the maximum current response elicited by a step to +10 mV. C The normalized current-voltage plot for an average of 272 cells. A Boltzmann equation fit revealed a V0.5 of activation of -4.8 mV.

Rundown of current is a phenomenon common to VGCC channels15 and can be a real problem for electrophysio-logical measurements. Using the perforated patch mode and multi-hole chips (4 holes per well) we could reliably record CaV1.2-mediated currents for >25 min (Figure 3).

CaV1.2 currents were blocked by nifedipine and verapamil in a concentration-dependent manner. The average concentration response curves are shown in Figure 4 and the IC50 values summarized in Table 1. The estimated IC50 values agree well with those found in the literature7-11.

Figure 3: Recordings of CaV1.2 channels expressed in CHO cells on the SyncroPatch® 384PE are stable over time. Shown are online analysis values peak amplitude versus time for 16 example cells. The recordings are stable for >25 min. The inset shows an enlarged graph.

Figure 4: Average concentration response curves for 2 different calcium channel blockers on the SyncroPatch® 384PE. Cumulative concentration response curves were performed and the concentration response curves were constructed and averaged for 8 columns of the NPC-384 plate as shown in Figure 5. The SyncroPatch® 384PE analysis software (DataControl® 384) was used to calculate the average concentration response curves. The points were normalized to control and fitted with a standard Hill-equation with minimum and maximum fixed to 0 and 1, respectively. A summary of the IC50 values and success rates is shown in Table 1.

A B

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phone +49 89 218997972fax +49 89 218997960www.nanion.de • info@nanion.de

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch® 384PE. Screenshot of depiction of online analysis data of CaV1.2 expressing CHO cells as recorded on one NPC-384 patch clamp chip (multi-hole, 4X). Control (0.3% DMSO), nifedipine or verapamil in increasing concentrations was applied to eight columns for each compound. Three hundred and eighty-four small color-coded pictures as seen in the upper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 50 MΩ), blue (Rmemb = 10 – 50 MΩ), light blue or grey (Rmemb < 10 MΩ or wells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show current amplitudes of CaV1.2 channels at the test pulse to 10 mV during application of reference solution (white region), and 3 applications of control (0.3% DMSO) or 3 different concentrations of nifedipine or verapamil. CaV1.2-mediated currents were stable over the entire experiment which lasted over 20 min.

Figure 6: Timeline of an experiment on the SyncroPatch® 384PE. The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) for a cumulative (3 concentrations per well) concentration response curve on CaV1.2-mediated currents took approximately 27 min. Each concentration of compound was incubated for approximately 4 min.

2 min

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5 min 20 min

Exp end and chip unload

Chip load and priming

Patch clamp experiment: 2 control washes followed by 3 compound additions

Analysis

Nanion Technologies GmbHGabrielenstr. 980636 Munich, Germany

phone +49 89 218997972fax +49 89 218997960www.nanion.de • info@nanion.de

Application Note

References1. Hofmann, F., et al. 2014. Physiol. Rev. 94: 303–326

2. Welling, A., et al., 1993. J.Physiol. 471: 749-765

3. Schultz, D., et al., 1993. PNAS. 90: 6228-6232

4. Napolitano, C. & Antzelevitch, C. 2011. Circ. Res. 108: 607-618

5. Sager, P., et al., 2014. Am. Heart J. 167: 292-300

6. Colatsky, T., et al., 2016. J. Pharmacol. Toxicol. Meth. 81: 15-20

7. Pignier, C. & Potreau, D. 2000. Am. J. Physiol. Heart Circ. Physiol. 279(5): H2259-2268

8. Liao, P,. et al., 2007. JBC. 282: 35133-35142

9. Kang, J., et al., 2012. JPET. 341(2): 510-517

10. Kuryshev, YA., et al., 2014 Assay Drug Dev. Tech. 12(2): 110-119

11. Cai, D., et al. 1997. Mol. Pharm. 51: 872–881

12. Mewes, T. & Ravens, U. 1994. J. Mol. Cell. Cardiol. 26 (10): 1307-1320

13. Bénitah, J.P. et al, 1992. Pflügers Arch. 421: 176-187

14. Belzmann, B. et al, 1998. Cardiovasc. Res. 38: 424-432

15. Belles, B., et al. 1988. Pflügers Arch. 1988. 411: 353 - 360

MethodsCellsCHO cells expressing CaV1.2/β2/α2δ1 provided by Charles River, Catalog #CT6004 .

Cell cultureCells were cultured and harvested according to Nanion’s standard cell culture protocol.

ElectrophysiologyPerforated patch clamp recordings were conducted according to Nanion’s standard procedure for the SyncroPatch® 384PE using multi-hole (4X) chips. The current-voltage relationship was elicited by depolarizing steps from -60 mV to 40 mV for 200 ms in 10 mV increments from a holding potential of -80 mV. A voltage step protocol from -80 mV (holding potential) to 10 mV for 200 ms was applied to the cells every 20 s for pharmacology experiments.

Compound IC50 Success rate (%)

Literature range

Nifedipine 12 nM (94) 74 9 - 220 nM7-10

Verapamil 7.6 µM (92) 72 0.17 - 110 µM9-11

Table 1: IC50 values for nifedipine and verapamil on CaV1.2- mediated currents recorded on the SyncroPatch® 384PE. Shown are IC50 values (number of cells shown in brackets), success rate for completed experiments and the expected literature IC50 values. IC50 values recorded on the SyncroPatch® 384PE agree well with the literature values7-11. Note that 1/3 of the plate received each compound, i.e. 128 wells.

Figure 5 shows a screenshot of the SyncroPatch® 384 software during an experiment. A color-coded over-view (based on seal resistance in this case) of all 384 wells gives the user a good impression of the success rate of the experiment. The user can choose whether to visualize raw traces or online analysis. Here, online analysis is chosen and the graphs represent current amplitude versus time. An individual well can be highlighted to monitor progression of the experiment. In the Online Analysis view, the time points at which solution additions have been made are indicated by vertical lines, as well as different background colors. In this example, white shows incubation in control

solution and blue is vehicle (0.3% DMSO) applied 3 times. In conclusion, CaV1.2 expressed in CHO cells can be recorded on the SyncroPatch® 384PE with high success rates for completed experiments (typically >70%). The timeline of each experiment was about 27 minutes (start – end) and included wash with vehicle followed by incubation in 3 concentrations of blocker (or control solution). Importantly, CaV1.2 displayed little or no rundown and current-voltage relationship recorded on the SyncroPatch® 384PE is in good agreement with the literature12-14. The IC50’s calculated using the Syncro- Patch® 384PE’s analysis software, DataControl® 384, of two calcium channel blockers were in good agreement with the literature7-11.

The SyncroPatch® 384PE is a high throughput and highly reliable automated patch clamp device for recording CaV1.2 currents. User-friendly software, excellent success rates, single additions or multiple additions of compound to each cell and easy analysis result in reliable high quality data at an increased throughput with an economical cost per data point.